Article

Multiple Forms of “Kiss-and-Run” Exocytosis Revealed by Evanescent Wave Microscopy

Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, BS8 1TD, Bristol, United Kingdom.
Current Biology (Impact Factor: 9.57). 05/2003; 13(7):563-7. DOI: 10.1016/S0960-9822(03)00176-3
Source: PubMed
ABSTRACT
Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that "kiss-and-run" mechanisms may also play a role. Here, we have examined the dynamics of exocytosis in pancreatic MIN6 beta cells by imaging lumen- (neuropeptide Y/pH-insensitive yellow fluorescent protein; NPY.Venus) or vesicle membrane-targeted fluorescent probes (synaptobrevin-2/enhanced green fluorescent protein; synapto.pHluorin, or phosphatase on the granule of insulinoma-enhanced green fluorescent protein, phogrin.EGFP) by evanescent wave microscopy. Unexpectedly, NPY.Venus release events occurred much less frequently (13%-40% maximal rate) than those of synapto.pHluorin, even though the latter molecule, but not phogrin.EGFP, usually diffused away from the site of fusion. Thus, the majority of exocytosis occurs in these cells by kiss-and-run events that involve either the release of small molecules only, small molecules and selected membrane proteins, or all soluble cargoes ("pure," "mixed," and "full" kiss-and-run, respectively). Changes in the activity of synaptotagmin IV, achieved here by overexpression of the wild-type protein, may allow different stimuli to alter the ratio of these events, and thus the release of selected vesicle cargoes.

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    • "Green/Yellow FP's with pKa>6 such as EGFP (pKa = 6) and pHluorin (pKa = 7.6) detect the luminal pH change during exocytosis and are therefore powerful tools to study membrane fusion and fission [9,10,24,30,34,66]. In (neuro)endocrine cells, dense core granules are large and sparse enough to be imaged individually and allow pH-sensitive FP's to be used to detect the opening of the exocytotic fusion pore and to measure its life time [9,19,35,61]. Since EGFP retains some of its fluorescence in intact granules, it is a preferred granule marker as it allows observation and tracking of the intact organelles before exocytosis. "
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    • "With a pK a (i.e., the pH value at which the fluorescence intensity is 50% of maximal) of 7.2, SEP is nearly nonfluorescent at pH 5.5 (the pH of intracellular secretory vesicles and recycling endosomes) but brightly green fluorescent at pH 7.4 (the extracellular pH). The SEP protein, fused to relevant membrane proteins, has been extensively used to detect the exocytosis of synaptic vesicles, secretory vesicles, and recycling endosomes (Sankaranarayanan et al., 2000; Gandhi and Stevens, 2003; Tsuboi and Rutter, 2003; Yudowski et al., 2006; Balaji and Ryan, 2007; Jullié et al., 2014). Moreover, it has been used to detect the formation of clathrin-coated vesicles (CCVs) using the pulsed pH (ppH) protocol (i.e., alternating the extracellular pH between 7.4 and 5.5), which reveals the location of receptors that have been newly internalized with high temporal accuracy (Merrifield et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation.
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    • "This analysis indicates that the calculated probability (∼2%) is much lower than the observed frequency (>10%,Figure 3G), suggesting that consecutive events are not randomly distributed events. Because fluorescence at the insertion site between consecutive events did not reach background levels (Figures 3B,D,F), we hypothesized that the fusion pore could close after an initial release of cargo and reopen for a consecutive release via a mechanism reminiscent of synaptic kiss-and-run (Stevens and Williams, 2000; Tsuboi and Rutter, 2003; Jaiswal et al., 2009; Alabi and Tsien, 2012). To test this hypothesis, we used the proton pump inhibitor bafilomycin to prevent reacidification of the closing vesicle after the initial release. "
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